Project Summary We currently lack effective therapies that reduce the risk of venous thromboembolism without an attendant risk of bleeding. The assembled investigative team has recently identified single chain antibodies that selectively block activated platelets and enrich therapeutics at the site of a developing thrombus. In the process, we have discovered that selective targeting of activated platelets allows a wide variety of antithrombotic agents to be efficacious, well below systemic concentrations that cause bleeding. In this proposal, we postulate that selective targeting to activated platelets provides a means to locally concentrate antiplatelet, anticoagulant, and fibrinolytic agents at the site of a growing venous thrombus without disrupting essential hemostatic processes. We believe that this strategy will enable the design of high potency agents for venous thromboprophylaxis, but without an increased risk of major bleeding. In this proposal, we intend to: (1) Define the ability of clot-targeted ?dual pathway? antithrombotics to prevent venous thrombosis and inhibit postthrombotic vein wall fibrosis without hemostatic disturbance. The efficacy of ?dual pathway? antithrombotics based on SCE5-TAP, a single chain antibody with both antiplatelet and anticoagulant activity will be defined using murine models of venous thrombosis. These studies will evaluate the capacity of SCE5-TAP and related new variants to prevent venous thrombosis, limit early and late inflammatory responses, and inhibit thrombus-induced remodeling of the vein wall without hemostatic disturbance. (2) Determine the capacity of clot-targeted antithrombotics that display anti-platelet and fibrinolytic activity to inhibit venous thrombosis and postthrombotic vein wall remodeling. The effectiveness of antithrombotics based on SCE5-scuPA, a single chain antibody with both antiplatelet and fibrinolytic activity will be defined using murine models of venous thrombosis alone or in combination with SCE5-TAP or related new variants. We will also design recombinant clot-targeted hybrid constructs (SCE5-TAP-scuPA) that display antiplatelet, anticoagulant, and fibrinolytic activity. The ability of these agents to inhibit thrombosis, promote thrombus resolution, and limit the development of vein wall fibrosis will be defined. (3) Mitigate the immune response to clot-targeted antithrombotics with the design of functionally deimmunized TAP variants through deletion of immunogenic T cell epitopes. The cellular immunogenicity of TAP (tick anticoagulant peptide) will be assessed and T cell epitopes, which drive the response mapped in nave human peripheral blood mononuclear cells. Computational tools will be used to design functionally deimmunized TAP variants that will be evaluated in vitro and ex vivo for their maintenance of FXa inhibitory activity and their reduction of cellular immunogenicity. The wild type and resulting lead candidate will be assessed for cellular and humoral immunogenicity in vivo in humanized HLA transgenic mice. The successful deimmunization of TAP will allow us to generate deimmunized clot-targeted antithrombotics.